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Monitoring Structural Transitions in IDPs by Vibrational Spectroscopy of Cyanylated Cysteine

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Intrinsically Disordered Protein Analysis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 895))

Abstract

The fast intrinsic time scale of infrared absorption and the sensitivity of molecular vibrational frequencies to their environments can be applied with site-specificity by introducing the artificial amino acid β-thiocyanatoalanine, or cyanylated cysteine, into chosen sites within intrinsically disordered proteins. This amino acid can be inserted through native chemical ligation at single cysteines introduced via site-directed mutagenesis. The CN stretching band of cyanylated cysteine is sensitive to local changes in both structural content and solvent exposure. This dual sensitivity makes cyanylated cysteine an especially useful probe of binding-induced structural transitions in IDPs. The general strategy of creating single-site cysteine mutations and chemically modifying them to create the vibrational chromophore, as well as observation, processing and analysis of the CN stretching band, is presented.

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References

  1. Bischak CG, Longhi S, Snead DM, Constanzo S, Terrer E, Londergan CH (2010) Probing structural transitions in the intrinsically disordered C-terminal domain of the measles virus nucleoprotein by vibrational spectroscopy of cyanylated cysteines. Biophys J 99:1676–1683

    Article  PubMed  CAS  Google Scholar 

  2. Choi JH, Oh KI, Cho MH (2008) Azido-derivatized compounds as IR probes of local electrostatic environment: theoretical studies. J Chem Phys 129:11

    Article  Google Scholar 

  3. Cremeens ME, Fujisaki H, Zhang Y, Zimmermann J, Sagle LB, Matsuda S, Dawson PE, Straub JE, Romesberg FE (2006) Efforts toward developing direct probes of protein dynamics. J Am Chem Soc 128:6028–6029

    Article  PubMed  CAS  Google Scholar 

  4. Fafarman AT, Webb LJ, Chuang JI, Boxer SG (2006) Site-specific conversion of cysteine thiols into thiocyanate creates an IR probe for electric fields in proteins. J Am Chem Soc 128:13356–13357

    Article  PubMed  CAS  Google Scholar 

  5. Getahun Z, Huang CY, Wang T, De Leon B, DeGrado WF, Gai F (2003) Using nitrile-derivatized amino acids as infrared probes of local environment. J Am Chem Soc 125:405–411

    Article  PubMed  CAS  Google Scholar 

  6. Taskent-Sezgin H, Chung JA, Banerjee PS, Nagarajan S, Dyer RB, Carrico I, Raleigh DP (2010) Azidohomoalanine: a conformationally sensitive IR probe of protein folding, protein structure, and electrostatics. Angew Chem Int Edit 49:7473–7475

    Article  CAS  Google Scholar 

  7. Waegele MM, Tucker MJ, Gai F (2009) 5-Cyanotryptophan as an infrared probe of local hydration status of proteins. Chem Phys Lett 478:249–253

    Article  PubMed  CAS  Google Scholar 

  8. Sagle LB, Zimmermann J, Dawson PE, Romesberg FE (2004) A high-resolution probe of protein folding. J Am Chem Soc 126:3384–3385

    Article  PubMed  CAS  Google Scholar 

  9. Schultz KC, Supekova L, Ryu YH, Xie JM, Perera R, Schultz PG (2006) A genetically encoded infrared probe. J Am Chem Soc 128:13984–13985

    Article  PubMed  CAS  Google Scholar 

  10. Ye SX, Huber T, Vogel R, Sakmar TP (2009) FTIR analysis of GPCR activation using azido probes. Nat Chem Biol 5:397–399

    Article  PubMed  CAS  Google Scholar 

  11. Degani Y, Degani C (1980) Enzymes with asymmetrically arranged subunits. Trends Biochem Sci 5:337–341

    Article  CAS  Google Scholar 

  12. Edelstein L, Stetz MA, McMahon HA, Londergan CH (2010) The effects of alpha-helical structure and cyanylated cysteine on each other. J Phys Chem B 114:4931–4936

    Article  PubMed  CAS  Google Scholar 

  13. Maienschein-Cline MG, Londergan CH (2007) The CN stretching band of aliphatic thiocyanate is sensitive to solvent dynamics and specific solvation. J Phys Chem A 111:10020–10025

    Article  PubMed  CAS  Google Scholar 

  14. He B, Wang K, Liu Y, Xue B, Uversky VN, Dunker AK (2009) Predicting intrinsic disorder in proteins: an overview. Cell Res. doi:10.1038/cr.2009.87

    Google Scholar 

  15. McMahon HA, Alfieri KN, Clark KAA, Londergan CH (2010) Cyanylated cysteine: a covalently attached vibrational probe of protein-lipid contacts. J Phys Chem Lett 1:850–855

    Article  PubMed  CAS  Google Scholar 

  16. Fuxreiter M, Tompa P, Simon I (2007) Local structural disorder imparts plasticity on linear motifs. Bioinformatics 23:950–956

    Article  PubMed  CAS  Google Scholar 

  17. Mohan A, Oldfield CJ, Radivojac P, Vacic V, Cortese MS, Dunker AK, Uversky VN (2006) Analysis of molecular recognition features (MoRFs). J Mol Biol 362:1043–1059

    Article  PubMed  CAS  Google Scholar 

  18. Oldfield CJ, Cheng Y, Cortese MS, Romero P, Uversky VN, Dunker AK (2005) Coupled folding and binding with alpha-helix-forming molecular recognition elements. Biochemistry 44:12454–12470

    Article  PubMed  CAS  Google Scholar 

  19. Vacic V, Oldfield CJ, Mohan A, Radivojac P, Cortese MS, Uversky VN, Dunker AK (2007) Characterization of molecular recognition features, MoRFs, and their binding partners. J Proteome Res 6:2351–2366

    Article  PubMed  CAS  Google Scholar 

  20. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratoty manual, 2nd edn. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  21. Benoit I, Coutard B, Oubelaid R, Asther M, Bignon C (2007) Expression in Escherichia coli, refolding and crystallization of Aspergillus niger feruloyl esterase A using a serial factorial approach. Protein Expr Purif 55:166–174

    Article  PubMed  CAS  Google Scholar 

  22. Lichty JJ, Malecki JL, Agnew HD, Michelson-Horowitz DJ, Tan S (2005) Comparison of affinity tags for protein purification. Protein Expr Purif 41:98–105

    Article  PubMed  CAS  Google Scholar 

  23. Hua QX, Jia WH, Bullock BP, Habener JF, Weiss MA (1998) Transcriptional activator-coactivator recognition: nascent folding of a kinase-inducible transactivation domain predicts its structure on coactivator binding. Biochemistry 37:5858–5866

    Article  PubMed  CAS  Google Scholar 

  24. Van Hoy M, Leuther KK, Kodadek T, Johnston SA (1993) The acidic activation domains of the GCN4 and GAL4 proteins are not alpha helical but form beta sheets. Cell 72:587–594

    Article  PubMed  Google Scholar 

  25. Whitmore L, Wallace BA (2004) DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res 32:W668–W673

    Article  PubMed  CAS  Google Scholar 

  26. Whitmore L, Wallace BA (2008) Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases. Biopolymers 89:392–400

    Article  PubMed  CAS  Google Scholar 

  27. Doherty GM, Motherway R, Mayhew SG, Malthouse JP (1992) 13C NMR of cyanylated flavodoxin from Megasphaera elsdenii and of thiocyanate model compounds. Biochemistry 31:7922–7930

    Article  PubMed  CAS  Google Scholar 

  28. Habchi J, Mamelli L, Darbon H, Longhi S (2010) Structural disorder within henipavirus nucleoprotein and phosphoprotein: from predictions to experimental assessment. PLoS One 5. doi:10.1371/journal.pone.0011684

    Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Chemistry, Haverford College, Haverford, PA, USA

    Hailiu Yang & Casey H. Londergan

  2. Architecture et Fonction des Macromolécules Biologiques, UMR 7257 CNRS and Aix-Marseille Université, Marseille, France

    Johnny Habchi & Sonia Longhi

Authors
  1. Hailiu Yang
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  2. Johnny Habchi
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  3. Sonia Longhi
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  4. Casey H. Londergan
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Corresponding author

Correspondence to Sonia Longhi .

Editor information

Editors and Affiliations

  1. Department of Molecular Medicine, College of Medicine, University of South Florida, Bruce B. Downs Blvd 12901, Tampa, 33612, Florida, USA

    Vladimir N. Uversky

  2. Dept. Biochemistry & Molecular Biology, Center for Computational Biology &, Indiana University, West 10th Street 410, Indianapolis, 46202, Indiana, USA

    A. Keith Dunker

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Yang, H., Habchi, J., Longhi, S., Londergan, C.H. (2012). Monitoring Structural Transitions in IDPs by Vibrational Spectroscopy of Cyanylated Cysteine. In: Uversky, V., Dunker, A. (eds) Intrinsically Disordered Protein Analysis. Methods in Molecular Biology, vol 895. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-927-3_17

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  • DOI: https://doi.org/10.1007/978-1-61779-927-3_17

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-926-6

  • Online ISBN: 978-1-61779-927-3

  • eBook Packages: Springer Protocols

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